Bottom Line:
Therapeutic advances do not circumvent the devastating fact that the survival rate in glioblastoma multiforme (GBM) is less than 5%.The in vitro studies revealed significantly higher binding of α -hEGFR-ILs when compared with liposomes conjugated with isotypic nonimmune immunoglobulin.The data show that α -hEGFR-ILs significantly enhance the uptake and accumulation of liposomes in this experimental model of GBM suggestive of improved specific nanoparticle-based delivery.

ABSTRACTTherapeutic advances do not circumvent the devastating fact that the survival rate in glioblastoma multiforme (GBM) is less than 5%. Nanoparticles consisting of liposome-based therapeutics are provided against a variety of cancer types including GBM, but available liposomal formulations are provided without targeting moieties, which increases the dosing demands to reach therapeutic concentrations with risks of side effects. We prepared PEGylated immunoliposomes (ILs) conjugated with anti-human epidermal growth factor receptor (EGFR) antibodies Cetuximab ( α -hEGFR-ILs). The affinity of the α -hEGFR-ILs for the EGF receptor was evaluated in vitro using U87 mg and U251 mg cells and in vivo using an intracranial U87 mg xenograft model. The xenograft model was additionally analyzed with respect to permeability to endogenous albumin, tumor size, and vascularization. The in vitro studies revealed significantly higher binding of α -hEGFR-ILs when compared with liposomes conjugated with isotypic nonimmune immunoglobulin. The uptake and internalization of the α -hEGFR-ILs by U87 mg cells were further confirmed by 3D deconvolution analyses. In vivo, the α -hEGFR-ILs accumulated to a higher extent inside the tumor when compared to nonimmune liposomes. The data show that α -hEGFR-ILs significantly enhance the uptake and accumulation of liposomes in this experimental model of GBM suggestive of improved specific nanoparticle-based delivery.

fig5: (A), Immunohistochemical characterization of the U87 mg induced intracranial tumor with regard to vascular density as detected by laminin-immunohistochemistry (A, T1) and permeability of albumin (T2). The density of capillaries is clearly higher within the tumor (T) marked with an asterisk compared to that of an area unaffected by tumor formation (N). (N2), (T2) Albumin-immunoreactivity (red) is present within the tumor indicative of a permeable vasculature, whereas albumin is seen occasionally only within the lumen of the brain capillaries. (N3), (T3) Overlays showing that endogenous albumin is present in the interstitium of mouse brain tumor tissue (T3), which contrasts that of the normal brain (N3). Cellular nuclei are visualized DAPI. Scale bar = 50 μm (N1–T3), 1 mm (A).

Mentions:
The tumor formation was examined macroscopically and verified by fluorescence microscopy in cryosections of the mouse brain injected with U87 mg cells (Figure 5). To access the vasculature, an immunohistochemical profile was performed to detect laminin of the basal membrane and endogenous plasma albumin as a marker of permeability (Figure 5). The vasculature between the normal brain and the tumor differed significantly. Hence, the vessels of the tumor were denser, larger in diameter, and overall very irregular compared with those of normal brain vessels (compare Figure 5(N1) with Figure 5(T1)). Endogenous mouse albumin was observed to accumulate extensively in the tumor interstitium indicative of higher permeability to macromolecules whereas, in the normal mouse brain, albumin was only confined to the vessels without appearance in the brain parenchyma indicative of an intact blood-brain barrier (compare Figures 5(N2) and 5(T2)).

fig5: (A), Immunohistochemical characterization of the U87 mg induced intracranial tumor with regard to vascular density as detected by laminin-immunohistochemistry (A, T1) and permeability of albumin (T2). The density of capillaries is clearly higher within the tumor (T) marked with an asterisk compared to that of an area unaffected by tumor formation (N). (N2), (T2) Albumin-immunoreactivity (red) is present within the tumor indicative of a permeable vasculature, whereas albumin is seen occasionally only within the lumen of the brain capillaries. (N3), (T3) Overlays showing that endogenous albumin is present in the interstitium of mouse brain tumor tissue (T3), which contrasts that of the normal brain (N3). Cellular nuclei are visualized DAPI. Scale bar = 50 μm (N1–T3), 1 mm (A).

Mentions:
The tumor formation was examined macroscopically and verified by fluorescence microscopy in cryosections of the mouse brain injected with U87 mg cells (Figure 5). To access the vasculature, an immunohistochemical profile was performed to detect laminin of the basal membrane and endogenous plasma albumin as a marker of permeability (Figure 5). The vasculature between the normal brain and the tumor differed significantly. Hence, the vessels of the tumor were denser, larger in diameter, and overall very irregular compared with those of normal brain vessels (compare Figure 5(N1) with Figure 5(T1)). Endogenous mouse albumin was observed to accumulate extensively in the tumor interstitium indicative of higher permeability to macromolecules whereas, in the normal mouse brain, albumin was only confined to the vessels without appearance in the brain parenchyma indicative of an intact blood-brain barrier (compare Figures 5(N2) and 5(T2)).

Bottom Line:
Therapeutic advances do not circumvent the devastating fact that the survival rate in glioblastoma multiforme (GBM) is less than 5%.The in vitro studies revealed significantly higher binding of α -hEGFR-ILs when compared with liposomes conjugated with isotypic nonimmune immunoglobulin.The data show that α -hEGFR-ILs significantly enhance the uptake and accumulation of liposomes in this experimental model of GBM suggestive of improved specific nanoparticle-based delivery.

ABSTRACTTherapeutic advances do not circumvent the devastating fact that the survival rate in glioblastoma multiforme (GBM) is less than 5%. Nanoparticles consisting of liposome-based therapeutics are provided against a variety of cancer types including GBM, but available liposomal formulations are provided without targeting moieties, which increases the dosing demands to reach therapeutic concentrations with risks of side effects. We prepared PEGylated immunoliposomes (ILs) conjugated with anti-human epidermal growth factor receptor (EGFR) antibodies Cetuximab ( α -hEGFR-ILs). The affinity of the α -hEGFR-ILs for the EGF receptor was evaluated in vitro using U87 mg and U251 mg cells and in vivo using an intracranial U87 mg xenograft model. The xenograft model was additionally analyzed with respect to permeability to endogenous albumin, tumor size, and vascularization. The in vitro studies revealed significantly higher binding of α -hEGFR-ILs when compared with liposomes conjugated with isotypic nonimmune immunoglobulin. The uptake and internalization of the α -hEGFR-ILs by U87 mg cells were further confirmed by 3D deconvolution analyses. In vivo, the α -hEGFR-ILs accumulated to a higher extent inside the tumor when compared to nonimmune liposomes. The data show that α -hEGFR-ILs significantly enhance the uptake and accumulation of liposomes in this experimental model of GBM suggestive of improved specific nanoparticle-based delivery.